If the gene is similar to RRM2, there is a likely chance that the gene has a similar expression profile and characteristics that would allow for a similar outcome of the gene being involved in the DNA repair pathway. However, it could still be possible that the gene might have a few different characteristics from the RRM2 gene that would cause for a different outcome where the gene wouldn’t be involved in the DNA repair pathway.
A gene expression profile is a way to analyze the expression of a specific gene at different times in a cell. The spikes at C4-C6 and C-14 are important in this research because they are points of conjugation in the cell, which requires DNA damage and repair. This is important for our gene because in the RRM2 expression profile there are spikes in expression of our gene during these points where DNA damage and repair occur, meaning there is a possibility that our gene could be involved in the DNA repair pathway.
We were able to assume that the primer designed for this experiment did effectively work and amplify our gene of interest because a band was produced around 484 base pairs, which was our expected result. The error within the experiment was in our positive control with the RPPO house keeping gene because there was no band produced when there should have been. Because of this we concluded that there was most likely a loading error when the gel electrophoresis was being done. The reason we hypothesized that our primers were not validated in this experiment was because of that issue with out positive control, so we could not completely trust the results that were produced, but there was no issue within the primers we designed.
I think that when completing the experiment again we would still use our validated primers and the designed primers because they were able to effectively amplify our gene correctly. For the next experiment I think that I would just need to be very specific and careful when performing it, because the issues in the experiments were mostly human error, such as loading a gel wrong or having the reagents in the PCR samples be nonfunctioning. Simple things that could be fixed in order to yield a result that allows for me to confidently say that our gene had increased expression when DNA damage was induced.
If another kind of gene that was similar to the R gene was used, do you think the outcomes would be similar or different?
If the gene is similar to RRM2, there is a likely chance that the gene has a similar expression profile and characteristics that would allow for a similar outcome of the gene being involved in the DNA repair pathway. However, it could still be possible that the gene might have a few different characteristics from the RRM2 gene that would cause for a different outcome where the gene wouldn’t be involved in the DNA repair pathway.
What is an expression profile and why are the spikes at C4-C6 and at C14 for the gene important in this case/what does it show?
A gene expression profile is a way to analyze the expression of a specific gene at different times in a cell. The spikes at C4-C6 and C-14 are important in this research because they are points of conjugation in the cell, which requires DNA damage and repair. This is important for our gene because in the RRM2 expression profile there are spikes in expression of our gene during these points where DNA damage and repair occur, meaning there is a possibility that our gene could be involved in the DNA repair pathway.
Do you have any hypotheses for why the primer you designed for your positive control ended up not working?
We were able to assume that the primer designed for this experiment did effectively work and amplify our gene of interest because a band was produced around 484 base pairs, which was our expected result. The error within the experiment was in our positive control with the RPPO house keeping gene because there was no band produced when there should have been. Because of this we concluded that there was most likely a loading error when the gel electrophoresis was being done. The reason we hypothesized that our primers were not validated in this experiment was because of that issue with out positive control, so we could not completely trust the results that were produced, but there was no issue within the primers we designed.
Do you have any ideas for why the primer you designed as a positive control ended up failing on the gel?
Is there anything you would do differently in your next experiment to ensure desired results?
I think that when completing the experiment again we would still use our validated primers and the designed primers because they were able to effectively amplify our gene correctly. For the next experiment I think that I would just need to be very specific and careful when performing it, because the issues in the experiments were mostly human error, such as loading a gel wrong or having the reagents in the PCR samples be nonfunctioning. Simple things that could be fixed in order to yield a result that allows for me to confidently say that our gene had increased expression when DNA damage was induced.